CN110152024A - A kind of ultrasound is with magnetic resonance bimodal targeted nano granule contrast agent and preparation method thereof - Google Patents
A kind of ultrasound is with magnetic resonance bimodal targeted nano granule contrast agent and preparation method thereof Download PDFInfo
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- CN110152024A CN110152024A CN201910487203.0A CN201910487203A CN110152024A CN 110152024 A CN110152024 A CN 110152024A CN 201910487203 A CN201910487203 A CN 201910487203A CN 110152024 A CN110152024 A CN 110152024A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1875—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle coated or functionalised with an antibody
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
Abstract
The present invention relates to molecular imagings and contrast-enhanced ultrasound technique field, more particularly to a kind of ultrasound and magnetic resonance bimodal targeted nano granule contrast agent, it includes lipidic shell and the pungent bromine alkane core of perfluor that is embedded in the lipidic shell, which is connected with anti-destination protein monoclonal antibody.The pungent bromine alkane of perfluor is the ingredient for playing ultrasound with magnetic resonance radiography function, anti- destination protein monoclonal antibody is the ingredient for playing the role of making contrast agent targeting destination organization or lesion, the pungent bromine alkane nanoparticle of perfluor and anti-destination protein monoclonal antibody bring out the best in each other, more easily target destination organization or lesion, after reaching destination organization or affected area concentration, ultrasonic development signal and magnetic resonance development signal are enhanced again, are realized and are developed to the ultrasound of destination organization or lesion with magnetic resonance bimodal.In particular by anti-CCR2 monoclonal antibody as anti-destination protein monoclonal antibody, can be used for monitoring myocardial ischemia-reperfusion injury.
Description
Technical field
The present invention relates to molecular imagings and contrast-enhanced ultrasound technique field, and in particular to a kind of ultrasonic and magnetic resonance bimodal
Targeted nano granule contrast agent and preparation method thereof, the targeted nano granule contrast agent are used especially for monitoring myocardial ischemia-reperfusion damage
Wound.
Background technique
In terms of ultrasound molecular imaging, have many inspirer research achievements both at home and abroad.However, traditional contrast agent
Still have the following problem when being applied to targeted ultrasound contrast: traditional acoustic contrast agent is using fluorocarbon gas as core
Microvesicle, since microvesicle partial size reaches micron order, relatively large, microvesicle is difficult to pass through blood vessel endothelium barrier；Acoustic resistive pressure energy power is poor,
Half-life period is shorter in body-internal-circulation, it is difficult to the requirement for needing certain targeting binding time when meeting targeted developing and observing repeatedly；
The background signal that the microvesicle of free state is formed in peripheral circulation when radiography is stronger, reduces " the noise of targeted developing enhancing
Than "；It can not achieve the multi-modal radiography of ultrasound/magnetic resonance, be unable to satisfy the demand of clinical a variety of image method integrated imagings.
The development of nano material and biotechnology haves a qualitative leap molecular imaging.The study found that with liquid fluorocarbon
For core material preparation lipid microsphere partial size up to nanoscale (Seo M, Williams R, Matsuura N.Size reduction
of cosolvent-infused microbubbles to form acoustically responsive
monodisperse perfluorocarbon nanodroplets.Lab Chip.2015；It 15:3581-3590), can be effective
Improve tissue penetration and the targeting efficiency of contrast agent in ground.It is cut moreover, liquid fluorocarbon particle has with traditional micro-bubble contrast agent
The sound reflecting signal that so different acoustics development mode, the i.e. contrast agent particle of free state generate is very faint, only when it
After assembling targeted to target cell and around it, the sound scattering phenomenon being remarkably reinforced just is generated, this will greatly improve target
Identified on the image when to radiography target cell signal-to-noise ratio (Xu X, Song R, He M, Peng C, Yu M, Hou Y, Qiu H,
Zou R,Yao S.A Microfluidic production of nanoscale perfluorocarbon droplets
as liquid contrast agents for ultrasound imaging.Lab Chip.2017；17:3504-3513).
However, existing liquid fluorocarbon is chiefly used in the ultrasonic contrast of single mode, there is not yet realizing ultrasound/magnetic resonance bimodal using it
The report of radiography.
Worldwide, ischemic heart disease is serious danger the main reason for leading to heart failure and then cause death
Evil human health.It is arranged at present clinically by thrombolysis, Coronary Artery Bypass Grafting and percutaneous coronary artery stent implantation etc.
It applies, narrow or even occlusion coronary artery lumen can be dredged, so as to improve the blood perfusion for even restoring cardiac muscle.However, the heart
Myocardial ischemia restores blood perfusion again after a certain period of time, and functional metabolism obstacle and the structure destruction of cardiac muscle cell does not mitigate not only instead
Aggravate, the phenomenon that ischemia injury is further aggravated after this blood reperfusion referred to as ischemical reperfusion injury, and then can lead to
Malignant arrhythmia occurs, left ventricular contraction power weakens, heart failure is even dead.Accurate measurements myocardial ischemia-reperfusion injury
And then corresponding clinical intervention measure is taken, it is played for reducing the Ischemic Heart Disease death rate, improving life in patients
Vital effect.Currently, clinically there is no the Imaging Method of effectively observation myocardial ischemia-reperfusion injury, heart muscle perfusion
There is certain limitation in imaging, myocardial metabolic imaging, interventional ultrasound imaging and mr imaging technique etc., clinically still
Consistency approval is not obtained.Therefore, study it is a kind of in real time and the Imaging Method of accurate measurements myocardial ischemia-reperfusion injury with
Just critical information is provided for clinical intervention decision and therapeutic effect assessment, is vital.
Studies have shown that inflammation plays very important effect in myocardial ischemia-reperfusion injury.Destemming after myocardial ischemia
Give up the idea outside flesh, there are still the cardiac muscle cells of survival, including Stunning myocardium and hibernating myocardium.Survival myocardium shows one kind and holds relatively
Long changes of function causes invertibity left chamber function to change.It is shown in animal cardiac muscle ischemia model, after restoring blood perfusion,
Suppression cardiac muscle and hibernating myocardium segment there is strong dynamic inflammatory reaction (Thind GS, Agrawal PR, Hirsh B,
Saravolatz L,Chen-Scarabelli C,Narula J,Scarabelli TM.Mechanisms of
myocardial ischemia-reperfusion injury and the cytoprotective role of
minocycline:scope and limitations.Future Cardiol.2015；11:61-76).This inflammatory reaction
Leukocyte infiltration based on the significantly high expression of monocyte chemoattractant protein-1 (MCP-1) and lasting monocyte is spy
Sign.MCP-1 is strong monocyte chemotactic inducer and activity factor, belongs to C-C subtribe (β subtribe) member.Study table
It is bright, in myocardial ischemic injury, the significant up-regulation of level of impaired cardiac muscle cell and microvascular endothelial cells secretion MCP-1
(Bae S,Park M,Kang C,Dilmen S,Kang TH,Kang DG,Ke Q,Lee SU,Lee D,Kang
PM.Hydrogen Peroxide-Responsive Nanoparticle Reduces Myocardial Ischemia/
Reperfusion Injury.J Am Heart Assoc.2016；5:e003697).CC class chemokine receptors 2 (CCR2) is
The specific receptor of MCP-1 belongs to g protein coupled receptor, there is seven " spiral transmembrane domains " structures for being rich in hydrophobic amino acid.Research
When showing to occur myocardial ischemia-reperfusion injury, the expression of impaired cardiac muscle cell and microvascular endothelial cells CCR2 are bright
It is aobvious to increase, and when impaired cardiac muscle cell is finally developed to the i.e. necrosis of irreversible damage, the inflammatory reaction of activation then tends to
It mitigates, leukocyte chemotaxis is reduced, and inflammatory factor synthesis reduces (Majmudar MD, Keliher EJ, Heidt T, et
al.Monocyte-directed RNAi targeting CCR2improves infarct healing in
atherosclerosis-prone mice.Circulation.2013；127(20):2038-2046).It is theoretical based on these,
It is concluded that CCR2 can be used as identification and monitor the promising target of myocardial ischemia-reperfusion injury.
In the diagnosis of heart disease, the various developing techniques such as ultrasound, magnetic resonance, CT are in spatial resolution, time resolution
The each tool advantage of rate etc., is used in combination to complement one another and provides diagnostic message more accurate and abundant for clinic.Therefore,
Multi-modal development has become the demand and trend of clinical diagnosis heart disease.The present inventor, which is conceived to, seriously endangers human health
Ischemic heart disease, trial prepare a kind of targeted contrast agent of specific recognition Myocardial Ischemia Reperfusion Injury, realize ultrasound/
The development of magnetic resonance bimodal, provide that more horn of plenty is comprehensive for clinical monitoring myocardial ischemia-reperfusion injury, more diagnostic value
Information.
Summary of the invention
The purpose of the present invention is being directed to the deficiency of existing acoustic contrast agent, and propose a kind of ultrasonic and magnetic resonance bimodal
Targeted nano granule contrast agent, which can be with targeted delivery to destination organization or lesion, and it is total with magnetic that ultrasound may be implemented
Shake bimodal radiography, is used especially for the ischemical reperfusion injury of monitoring cardiac muscular tissue.
The purpose of the present invention is achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of ultrasonic and magnetic resonance bimodal targeted nano granule contrast agent, which is received
Grain of rice contrast agent includes lipidic shell and the pungent bromine alkane core of perfluor that is embedded in the lipidic shell, which connects
It is connected to anti-destination protein monoclonal antibody.
In the ultrasound and magnetic resonance bimodal targeted nano granule contrast agent of first aspect present invention, the pungent bromine alkane of perfluor is hair
The ingredient of ultrasound with magnetic resonance radiography function is waved, anti-destination protein monoclonal antibody is to play that the contrast agent is made to target destination organization
Or the ingredient of the effect of lesion.
In the specific embodiment of first aspect present invention, which includes distearyl acid phosphatldylcholine, two
Stearic acid phosphatidyl ethanolamine-polyethylene glycol, distearoylphosphatidylglycerol and cholesterol.
In the specific embodiment of first aspect present invention, the distearyl acid phosphatldylcholine, distearyl acid phosphatidyl
The molar ratio of ethanol amine-polyethylene glycol, distearoylphosphatidylglycerol and cholesterol is 8.5:(0.5-1): (0.5-1): (0.5-
1).Preferably, the distearyl acid phosphatldylcholine, distearyl acid phosphatidyl ethanolamine-polyethylene glycol, distearoylphosphatidyl are sweet
The molar ratio of oil and cholesterol is 8:0.5:0.5:1.
In the specific embodiment of first aspect present invention, which passes through biotin-parent
The lipidic shell outer surface is connected to element-biotin bridging.
In the specific embodiment of first aspect present invention, which is that coupling has the first life
The anti-destination protein monoclonal antibody of the biotinylation of object element, the distearyl acid phosphatidyl ethanolamine-polyethylene glycol are wherein poly- second
Glycol moiety, which is coupled, distearyl acid phosphatidyl ethanolamine-polyethylene glycol-biotin of the second biotin, first biotin and
Second biotin is connected by Avidin, and the anti-destination protein monoclonal antibody is made to be connected to the lipidic shell outer surface.
In the specific embodiment of first aspect present invention, which is cetomacrogol 1000, polyethylene glycol
1500, polyethylene glycol 2000, Macrogol 4000 or Macrogol 6000.Preferably, which is polyethylene glycol 2000.
In the specific embodiment of first aspect present invention, which is CC class chemokine receptors 2 (CCR2),
The anti-destination protein monoclonal antibody is anti-CCR2 monoclonal antibody.
In second aspect, the present invention provide it is a kind of prepare ultrasound with magnetic resonance bimodal targeted nano granule radiography agent method,
Method includes the following steps:
(1) it is 8.5:(0.5-1 by molar ratio): (0.5-1): distearyl acid phosphatldylcholine, the distearyl acid of (0.5-1)
Phosphatidyl ethanolamine-polyethylene glycol-biotin, distearoylphosphatidylglycerol and cholesterol are added organic molten in reaction vessel
In agent, dissolution forms clear solution；
(2) it is evaporated in vacuo the clear solution, to form thin film in the reaction vessel；
(3) aqueous buffer is added in the reaction vessel, which is formed into mother liquor；
(4) the pungent bromine alkane of perfluor is added dropwise to the mother liquor under stiring, obtains mixed liquor；
(6) mixed liquor is added in liposome extruder, the nanopore membrane through the liposome extruder obtains biology
The pungent bromine alkane nanoparticle of elementization perfluor；
(7) excessive Avidin is added to the pungent bromine alkane nanoparticle of the biotinylation perfluor, is incubated for 30- at 0-4 DEG C
60min, centrifugation obtain the pungent bromine alkane nanoparticle-Avidin compound of biotinylation perfluor；
(8) the excessive anti-purpose of biotinylation is added to the pungent bromine alkane nanoparticle of the biotinylation perfluor-Avidin compound
Protein monoclonal antibody, is incubated for 30-60min at 0-4 DEG C, and centrifugation obtains targeting ultrasound and magnetic resonance pair of the destination protein
Mode targeted nano granule contrast agent.
It can according to need the nanometer micropore size for selecting the nanopore membrane of the liposome extruder, to prepare not
With the nanoparticle of nano-scale, for example, the average grain diameter of the pungent bromine alkane nanoparticle of biotinylation perfluor of the invention can be in 100-
Any numerical value in the range of 200nm, such as 100nm, 120nm, 150nm, 180nm, 200nm or between them.
In the specific embodiment of second aspect of the present invention, the distearyl acid phosphatldylcholine, distearyl acid phosphatidyl
Ethanol amine-polyethylene glycol-biotin, distearoylphosphatidylglycerol and cholesterol molar ratio is 8:0.5:0.5:1, this is organic
Solvent is chloroform, the aqueous buffer solution be phosphate buffer, the polyethylene glycol be cetomacrogol 1000, polyethylene glycol 1500,
Polyethylene glycol 2000, Macrogol 4000 or Macrogol 6000, it is preferable that the polyethylene glycol is polyethylene glycol 2000.
In the specific embodiment of second aspect of the present invention, which is CC class chemokine receptors 2 (CCR2),
The anti-destination protein monoclonal antibody is anti-CCR2 monoclonal antibody.
It should be noted that ultrasound of the invention and the pungent bromine of perfluor in magnetic resonance bimodal targeted nano granule contrast agent
The content of alkane is not particularly limited, as long as meeting the needs of radiography, but in general, the pungent bromine alkane of perfluor and shell lipid molecular
Molar ratio is between 1:10-1:30.
Beneficial effects of the present invention:
Ultrasound of the invention in magnetic resonance bimodal targeted nano granule contrast agent, the pungent bromine alkane of perfluor be play ultrasound with
The ingredient of magnetic resonance radiography function, anti-destination protein monoclonal antibody, which plays, makes contrast agent targeting destination organization or lesion
The ingredient of effect.
Ultrasound of the invention compared with traditional targeted contrast agent, has such as with magnetic resonance bimodal targeted nano granule contrast agent
Lower advantage: (1) partial size is smaller: the present inventor uses nano preparation technique, has successfully prepared the pungent bromine alkane nanoparticle of perfluor, grain
Diameter is smaller than traditional micro-bubble contrast agent, stronger, the more high advantage of stability with tissue penetration, more suitable for targeted ultrasound
Radiography；(2) there is stronger resistance to acoustic pressure: no matter in high mechanical index or low mechanical index sound field, with traditional micro-bubble
Contrast agent is compared, and the resistance to acoustic pressure of the pungent bromine alkane nanoparticle of perfluor is all more preferable, more suitable for targeted ultrasound contrast；(3) stability is high:
Targeted nano granule contrast agent of the present invention it is not easy to reunite, and surface tension is small, more stable；(4) ultrasound targeting radiography signal-to-noise ratio
Improve: the present inventor, which studies the discovery pungent bromine alkane nanoparticle of perfluor, has the ultrasonic development different from traditional micro-bubble contrast agent special
Sign, i.e. its echo signal for generating under free state are very faint, are shown as echoless, only after forming coherent condition,
Contrast agent signals are just remarkably reinforced, and this special ultrasonic development feature can have more advantage in targeting radiography, not will receive back
The interference of scape signal；(5) ultrasound-magnetic resonance bimodal development can be achieved: the pungent bromine alkane of perfluor contains the fluorine atom of high concentration, can incite somebody to action
It is applied to19F Magnetic Resonance Imaging (19F-MRI), it is total can to significantly increase magnetic under free state for the pungent bromine alkane nanoparticle of perfluor
Vibration development (MRI) signal, and signal enhancing becomes apparent under coherent condition.
Therefore, the pungent bromine alkane nanoparticle of perfluor of ultrasound of the invention and magnetic resonance bimodal targeted nano granule contrast agent and resist
Destination protein monoclonal antibody brings out the best in each other, it is easier to target destination organization or lesion, reach destination organization or affected area collection
In after, and enhance ultrasonic development signal and magnetic resonance development signal, realize to the ultrasound and magnetic resonance of destination organization or lesion
Bimodal.
As described in " background technique ", the iconography side of effectively observation myocardial ischemia-reperfusion injury clinically there is no at present
Method, the present inventor is using anti-CCR2 monoclonal antibody as in present invention ultrasound and magnetic resonance bimodal targeted nano granule contrast agent
Anti- destination protein monoclonal antibody, obtained targeted nano granule contrast agent can target Myocardial Ischemia-reperfusion Injury segment,
Specificity enhances the ultrasonic signal and magnetic resonance signal at the position, realizes multi-modal targeting radiography, to realize to myocardial ischemia
Sensitive, efficient, the noninvasive identification and monitoring of reperfusion injury.
Detailed description of the invention
Fig. 1 be display targeting CCR2 the pungent bromine alkane nanoparticle contrast agent of perfluor construction and its with the site CCR2 on target cell
Target the schematic diagram combined；
Fig. 2 is the pungent bromine alkane nanoparticle aspect graph of perfluor and grain size distribution, and wherein A is the pungent bromine alkane nanoparticle transmission electricity of perfluor
It is seen under mirror；B is the pungent bromine alkane nanoparticle grain size distribution of perfluor, and nanoparticle average grain diameter is 152.3nm；
Fig. 3 show the pungent bromine alkane nanoparticle of blank perfluor and the pungent bromine alkane nanoparticle of biotinylation perfluor be added before Avidin and
Wherein A: ultrasonic development situation later before Avidin is added in the pungent bromine alkane nanoparticle of blank perfluor, is shown as echoless；B: blank
After Avidin is added in the pungent bromine alkane nanoparticle of perfluor, echoless is still appeared as；C: parent is added in the pungent bromine alkane nanoparticle of biotinylation perfluor
Before element, it is shown as echoless；D: after Avidin is added in the pungent bromine alkane nanoparticle of biotinylation perfluor, echo is remarkably reinforced, display
For intensive tiny dotted strong echo；
Fig. 4 shows that the pungent bromine alkane nanoparticle of perfluor is in the development of different ultrasound wave irradiation times under high sound pressure environment (MI 0.56)
Situation, wherein A, B, C, D are respectively development situation of the pungent bromine alkane nanoparticle of perfluor after ultrasound wave irradiation 0s, 10s, 20s, 30s, not
See significant change；
Fig. 5 shows ischemia-reperfusion model rat heart short axis view, wherein A: the injection CCR2 targeting pungent bromine alkane of perfluor is received
Before the grain of rice；B: after the injection CCR2 targeting pungent bromine alkane nanoparticle of perfluor, see that myocardium echo is remarkably reinforced (white under the nearly inner membrance of antetheca
Shown in color arrow), and remaining locular wall has no and is remarkably reinforced；
Fig. 6 shows magnetic resonance development (MRI) situation of the pungent bromine alkane nanoparticle of perfluor, wherein A: deionized water；B: deionization
Water+Avidin；C: the pungent bromine alkane nanoparticle of biotinylation perfluor；The pungent bromine alkane nanoparticle+Avidin of D biotinylation perfluor；
Fig. 7 shows Ischemia and Reperfusion in vivo in Rats phantom heart frozen section fluorescence microscopy under the microscope (× 100).
Specific embodiment
Below by specific embodiment and in conjunction with attached drawing, invention is further described in detail.
As described in " summary of the invention " part above, the present invention provides a kind of ultrasound and makes with magnetic resonance bimodal targeted nano granule
Shadow agent, the targeted nano granule contrast agent include lipidic shell and the pungent bromine alkane core of perfluor that is embedded in the lipidic shell, the rouge
Matter outer surface of outer cover is connected with anti-destination protein monoclonal antibody.The lipidic shell includes distearyl acid phosphatldylcholine, two hard
Resin acid phosphatidyl ethanolamine-polyethylene glycol, distearoylphosphatidylglycerol and cholesterol.The anti-destination protein monoclonal antibody is logical
It crosses biotin-avidin-biotin bridging and is connected to the lipidic shell outer surface.Specifically, the anti-destination protein monoclonal antibody
To be coupled the anti-destination protein monoclonal antibody of biotinylation for having the first biotin, the distearyl acid phosphatidyl ethanolamine-poly- second
Glycol is distearyl acid phosphatidyl ethanolamine-polyethylene glycol-biotin that wherein polyalkylene glycol moiety coupling has the second biotin,
First biotin is connected with second biotin by Avidin, and the anti-destination protein monoclonal antibody is made to be connected to the lipid
Outer surface of outer cover.Specifically, which is CC class chemokine receptors 2 (CCR2), the anti-destination protein monoclonal antibody
For anti-CCR2 monoclonal antibody.
I. the pungent bromine alkane of perfluor
Traditional contrast agent is using fluorocarbon gas as kernel, although can be by adjusting ultrasonic cell disintegration instrument in sound and vibration step
Output power control its partial size, but effect and unstable is easy to be influenced by other factors；And since microvesicle is easily broken
It is bad that gas is caused to escape, it is also difficult to further by its size controlling in smaller size.
The present invention uses the pungent bromine (C of perfluor8F17Br) as the design of kernel.The pungent bromine alkane of perfluor is a kind of liquid fluorocarbon, is
Safe and non-toxic compound is clinically widely used as blood substitute earliest, with highly stable chemical characteristic and
Good oxygen carrier function.The boiling point of the pungent bromine alkane of perfluor is 142 DEG C, is at normal temperature liquid, not soluble in water, and property is stablized, and close
Larger (1.93g/ml) is spent, there is good anti-pressure ability, will not be destroyed under higher acoustic pressure environment, to make
Its development effect is maintained.
In the preparation process of the pungent bromine of perfluor, can be used the high shear force environments such as high speed dispersion homogenizer to its partial size into
Row control, finally can further be controlled the maintenance level within 200nm with liposome extruder.Particle size
It substantially reduces, is greatly improved the ability that targeted nano granule of the present invention penetrates vascular endothelial cell barrier.
Moreover, because the pungent bromine alkane of perfluor contains the fluorine atom of high concentration, 19F Magnetic Resonance Imaging (19F- can be applied to
MRI).19F has a characteristic that natural abundance is high (100%), has and the comparable sensitivity of 1H；Chemical shift distribution is wide；Absolutely
19F is all free of in most of organism, thus is interfered without background signal.
II. lipidic shell
Lipidic shell, which is used to for the pungent bromine alkane of perfluor being embedded in, wherein makes targeted nano granule contrast agent of the invention form core-
Shell structure.The ingredient of lipidic shell can for distearyl acid phosphatldylcholine, distearyl acid phosphatidyl ethanolamine-polyethylene glycol,
Distearoylphosphatidylglycerol and cholesterol, but these components are not limited to, the ingredient for preparing liposome commonly used in the art all may be used
To use.
It is connected to outside the lipidic shell in the anti-destination protein monoclonal antibody by biotin-avidin-biotin bridging
In the case where surface, distearyl acid phosphatidyl ethanolamine-polyethylene glycol is that wherein polyalkylene glycol moiety coupling has the two of biotin
Stearic acid phosphatidyl ethanolamine-polyethylene glycol-biotin.
Distearyl acid phosphatldylcholine, distearoylphosphatidylglycerol and the commercially available acquisition of cholesterol.Distearyl acid phosphorus
Ester acyl ethanol amine-polyethylene glycol and distearyl acid phosphatidyl ethanolamine-polyethylene glycol-biotin can be from U.S. Avanti Polar
Lipids companies market obtains.
Distearyl acid phosphatldylcholine is added in filmogen the present invention, and microbubble surface obtained is negatively charged, so that
Intergranular is mutually exclusive is not susceptible to reunite for nanometer.In addition, being C at nanoparticle contrast agent outer membrane of the present invention8F17What Br and water were formed
Liquid-liquid interface, and the surface tension of liquid-liquid interface is much smaller than the liquid-vapor interface of traditional contrast agent microbubble, makes the nanoparticle of preparation
Contrast agent more meets molecular mechanics requirement, forms a kind of highly stable system, is greatly improved targeted nano of the present invention
The stability of grain contrast agent.
III. destination protein, CCR-2
Anti- destination protein monoclonal antibody enables targeted nano granule contrast agent of the invention to target and is delivered to interested
Destination organization or lesion.Destination protein is the specific indicator protein of interested disease, is anti-destination protein monoclonal antibody
Target spot, targeted nano granule contrast agent of the invention can be guided to be delivered to interested destination organization or lesion, so as to this
Position carries out radiography.For example, in the present invention, destination protein can be CC class chemokine receptors 2 (CCR2).
CCR-2 is the specific receptor of MCP-1, belongs to g protein coupled receptor, there is seven " spirals for being rich in hydrophobic amino acid
Transmembrane domains " structure.Studies have shown that when myocardial ischemia-reperfusion injury occurs, impaired cardiac muscle cell and microvascular endothelial cells
The expression of CCR2 is significantly raised, and when impaired cardiac muscle cell is finally developed to the i.e. necrosis of irreversible damage, activation
Inflammatory reaction then tend to mitigate, leukocyte chemotaxis reduce, inflammatory factor synthesis reduce, it is inferred that CCR2 can be used as identification
And the promising target of monitoring myocardial ischemia-reperfusion injury.
Fig. 1 schematically illustrate targeting CCR2 the pungent bromine alkane nanoparticle contrast agent of perfluor construction and its on target cell
The targeting of the site CCR2 combines.
Further illustrate is made to the present invention below by way of non-limiting embodiment.
Embodiment 1: preparation, physicochemical property identification and the study on the stability of the pungent bromine alkane nanoparticle of perfluor
1. the preparation of the pungent bromine alkane nanoparticle of perfluor
Weigh distearyl acid phosphatldylcholine (DSPC), the distearyl acid phosphatidyl that molar ratio is 8.5:0.5:0.5:0.5
Ethanol amine polyethylene glycol 2000 (DSPE-PEG2000), distearoylphosphatidylglycerol (DSPG) and cholesterol (CH), are placed in circle
In the flask of bottom, it is molten to clarification that 15ml chloroform is added.The flask is placed on Rotary Evaporators, after vacuumizing evaporation 2h, forms one
The uniform film of layer.It takes the addition of 20ml PBS solution to be evaporated in the round-bottomed flask of film forming, after aquation 1h, obtains milky mother liquor.
Mother liquor is moved in beaker, then with high speed dispersion homogenizer, and the pungent bromine alkane of 0.2ml perfluor is added dropwise simultaneously.It is mixed by what is obtained
It closes liquid to be added in liposome extruder (LF-50, Canadian Northern Lipids company), cross 200nm micropore film process 3 times,
Obtain the pungent bromine alkane nanoparticle of perfluor.
2. the form and particle diameter distribution of the pungent bromine alkane nanoparticle of perfluor
By the pungent bromine alkane nanoparticle of obtained perfluor to be observed under transmission electron microscope after pure water dilutes 10 times in the form of it.Using swash
Light Particle Size Analyzer (ZETASIZER NANO ZS, Malvern company, Britain) measures the partial size point of the pungent bromine alkane nanoparticle of perfluor
Cloth.Fig. 2 shows the form and particle diameter distribution of the pungent bromine alkane nanoparticle of perfluor, and wherein Fig. 2A shows the pungent bromine alkane nanoparticle transmission of perfluor
It is seen under Electronic Speculum；Fig. 2 B shows that the pungent bromine alkane nanoparticle particle diameter distribution of perfluor, nanoparticle average grain diameter are 152.3nm.
The pungent bromine alkane nanoparticle of the perfluor of the present embodiment does not connect biotin and is coupled anti-destination protein monoclonal antibody, can
The referred to as pungent bromine alkane nanoparticle of blank perfluor.
The ultrasonic characteristic of the pungent bromine alkane nanoparticle of 2. perfluor of embodiment and19F-MRI development effect is investigated
1. the ultrasonic characteristic of the pungent bromine alkane nanoparticle of perfluor is investigated
The preparation method of reference implementation example 1, the pungent bromine alkane nanoparticle of preparation blank perfluor and the pungent bromine alkane of biotinylation perfluor are received
The grain of rice, material use DSPC, DSPE-PEG2000, DSPG and CH and DSPC, DSPE-PEG2000, biotinylation respectively
Distearyl acid phosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG2000-Biotin), DSPG and CH.
2ml is taken to be placed in 10ml EP respectively the pungent bromine alkane nanoparticle of blank perfluor and the pungent bromine alkane nanoparticle of biotinylation perfluor
Guan Zhong, using 15L8w-s high frequency probe (frequency 12.0MHz), observation development situation simultaneously acquires image.Respectively in two samples
0.5mg Avidin (concentration 0.25mg/ml) and then secondary its development effect of observation is added and acquires image.With Metlab
The image of image analysis software analysis acquisition, two groups of contrast agent of record are added before Avidins and average gray-scale value later, and
It is compared.The acoustic signal feature that the pungent bromine alkane nanoparticle of perfluor is observed under different ultrasonic energies, detects its resistance to acoustic pressure
Energy.
Fig. 3 shows that the pungent bromine alkane nanoparticle of blank perfluor and the pungent bromine alkane nanoparticle of biotinylation perfluor are added before Avidin
Ultrasonic development situation later.Before Fig. 3 A shows that Avidin is added in the pungent bromine alkane nanoparticle of blank perfluor, it is shown as echoless；Figure
After 3B shows that Avidin is added in the pungent bromine alkane nanoparticle of blank perfluor, echoless is still appeared as；Fig. 3 C shows that biotinylation perfluor is pungent
Before Avidin is added in bromine alkane nanoparticle, it is shown as echoless；It is affine that Fig. 3 D shows that the pungent bromine alkane nanoparticle of biotinylation perfluor is added
After element, echo is remarkably reinforced, and is shown as intensive tiny dotted strong echo.Fig. 3 demonstrates targeted nano granule radiography of the invention
Agent has good development effect and targeting radiography signal-to-noise ratio, and grayscale intensity is 1.91 ± .66, grayscale intensity when aggregation when dissociating
It is 82.19 ± 15.68.
Fig. 4 shows that the pungent bromine alkane nanoparticle of perfluor is in the development of different ultrasound wave irradiation times under high sound pressure environment (MI 0.56)
Situation, wherein A, B, C, D are respectively development situation of the pungent bromine alkane nanoparticle of perfluor after ultrasound wave irradiation 0s, 10s, 20s, 30s, not
See significant change, it was demonstrated that targeted nano granule contrast agent of the invention still has good resistance to acoustic pressure in high mechanical index sound field
Property.
2. the pungent bromine alkane nanoparticle of perfluor19F-MRI development effect is investigated
The pungent bromine alkane nanoparticle of perfluor is prepared as described in Example 1.20 healthy adult SD rats are randomly divided into 2 groups,
The pungent bromine alkane nanoparticle of perfluor and physiological saline are injected by tail vein respectively.Observe the liver of two groups of rats19F-MRI development feelings
Condition is analyzed two groups of hepatic parenchymal signal strengths of rat in work station, and is compared.
The preparation of the embodiment 3.CCR2 targeting pungent bromine alkane nanoparticle of perfluor
As described in Example 1, but with DSPE-PEG2000-Biotin DSPE-PEG2000 is substituted, prepares biotinylation
The pungent bromine alkane nanoparticle of perfluor.
Anti- CCR2 monoclonal antibody is connected to the pungent bromine of biotinylation perfluor using biotin-avidin-biotin bridging
On alkane nanoparticle, the specific method is as follows.Low-speed centrifugal abandons supernatant with the pungent bromine alkane nanoparticle (NP-B) of purifying biological element perfluor.
Excessive Avidin (AD) is added by nanoparticle concentration, 30min is incubated at 4 DEG C, centrifugation abandons supernatant collection lower layer and combines parent
With the nanoparticle (NP-B-AD) of element.The anti-CCR2 monoclonal antibody of excessive biotinylation is added into NP-B-AD solution, 4 DEG C
Lower incubation 30min, centrifugation abandon supernatant and collect the CCR2 targeting pungent bromine alkane of perfluor for combining the anti-CCR2 monoclonal antibody of biotinylation
Nanoparticle (NP-B-AD-B-CCR2).
Embodiment 4.CCR2 targets the external target-seeking efficiency evaluation of the pungent bromine alkane nanoparticle of perfluor
1. normal and anoxic myocardial culture
The suckling mouse apex tissue of 1-2d after taking-up is raw, pancreatin digestion, with the DMEM training containing 20% calf serum after centrifugation
Cell suspension is made in feeding base, filtering, purifies cardiac muscle cell by differential velocity adherent partition method, is inoculated in 24 orifice plates, per replacement culture for 24 hours
Liquid to get arrive normal myocardial cells.After adherent growth 2d, it is changed to plasma-free DMEM medium for 24 hours before experiment, random point
Group, every group of double parallel hole, is repeated 4 times operation.First by culture medium N21h or more is balanced in closed container, and anaerobic training is made
Base is supported, anaerobic culture medium is added after culture medium oxygen-containing in culture hole is sucked out, then culture plate is placed in containing 5%CO2- 95%N2
(V/V) anoxic is carried out in the closed container of mixed gas at 37 DEG C and is incubated for 10h to get anoxic myocardial is arrived.
2.CCR2 targets the external target-seeking efficiency evaluation of the pungent bromine alkane nanoparticle of perfluor
It prepares fluorescence nano grain: DSPC being substituted with the DSPC (NBD-DSPC) of fluorescent marker, is distinguished as described in Example 1
It prepares the pungent bromine alkane nanoparticle of blank control perfluor and CCR2 targets the pungent bromine alkane nanoparticle of perfluor.
Two groups of nanoparticles of preparation are added in the normal myocardial cells and anoxic myocardial culture dish of in vitro culture, 37
DEG C be incubated for 30min, PBS rinse 3 times, wash away the microvesicle not sticked.DAPI is added and carries out nuclear targeting.It is seen under fluorescence microscope
Examine the situation of sticking of microvesicle and cell, the microvesicle for calculating cell peripheral under 5 200 times of visuals field sticks number, and between each group into
Row compares.
Embodiment 5.CCR2 targets target-seeking efficiency evaluation in the pungent bromine alkane nanoparticle body of perfluor
1. the foundation of myocardial ischemia-reperfusion model in rats and model identification
Healthy adult Wistar rat abdominal cavity is anaesthetized, dorsal position is fixed on experimental bench, connects electrocardiograph, and tracheae is inserted
It manages and fixes, connect toy ventilator.Chest is opened, pericardium is removed, heart is sufficiently exposed, is sought between pulmonary conus and left auricle of heart
It looks for left anterior descending coronary artery (LAD) and ligatures.Observation dominates regional myocardial situation of change downstream, with regional myocardial presentation
There are ST sections of ischemics of typical case and change into ligation successfully mark in canescence and electrocardiogram.Ligature is cut after 30min, then fills the pass 2h
Chest is spare.Four modeling rats are randomly selected, its heart, the bis- dyeing of two row TTC-EB, another two row HE are taken after the completion of experiment
Microscopically observation is dyed, model is evaluated.Sham-operation control rats: rat anesthesia, trachea cannula, open after chest without
Ligation is handled, and it is spare that chest is closed after mechanical ventilation 2.5h.
2.CCR2 targets target-seeking efficiency evaluation in the pungent bromine alkane nanoparticle body of perfluor
It prepares fluorescence nano grain: DSPC being substituted with the DSPC (NBD-DSPC) of fluorescent marker, is distinguished as described in Example 1
It prepares the pungent bromine alkane nanoparticle of blank control perfluor and CCR2 targets the pungent bromine alkane nanoparticle of perfluor.
Experimental group: myocardial ischemia-reperfusion rat model and sham-operation control rats random number are injected after grouping
Nanoparticle carries out targeting radiography.Specific experiment is grouped as follows: the pungent bromine alkane nanoparticle group of (i) sham-operation rat+blank control perfluor:
Take 0.5ml in tail vein injection sham-operation rat body (bolus) the pungent bromine alkane nanoparticle of the blank control perfluor of preparation, it is rear to infuse
Enter 1ml PBS；(ii) sham-operation rat+CCR2 targets the pungent bromine alkane nanoparticle group of perfluor: the CCR2 of preparation is targeted the pungent bromine of perfluor
Alkane nanoparticle takes 0.5ml in tail vein injection sham-operation rat body (bolus), rear to inject 1ml PBS；(iii) ischemia-reperfusion
Rat model+blank control nanoparticle group: 0.5ml is taken to inject through tail vein the pungent bromine alkane nanoparticle of the blank control perfluor of preparation
It is rear to inject 1ml PBS in rat model body (bolus)；(iv) ischemia-reperfusion injury model rat+CCR2 targets the pungent bromine alkane of perfluor and receives
Grain of rice group: taking 0.5ml in tail vein injection model rat body (bolus) the CCR2 targeting pungent bromine alkane nanoparticle of perfluor of preparation,
1ml PBS is injected afterwards.
The ultrasonic targeted developing effect of each group is evaluated with compuscan and is compared: being intended that super with Vevo2100
The real-time molecular imaging system of sound, MS250 pops one's head in (center probe frequency 20MHz, power 10%, mechanical index 0.3), before injection
It observes rat heart image, dynamic observation myocardial visualization situation respectively afterwards, and acquires image.Later period is incidentally fixed using Vevo2100
Amount analysis software carries out later image analysis, measures each group rat antetheca, side wall, rear wall and the interventricular septum heart in left room short axis view
Flesh signal strength (grayscale value, GS) and signal-to-noise ratio (SNR) are simultaneously compared.The internal targeted developing of ultrasound is joined during the experiment
Number carries out reasonably optimizing.
Fig. 5 shows ultrasound observation ischemia-reperfusion model rat heart short axis view, and wherein A figure is injection CCR2 targeting
Before the pungent bromine alkane nanoparticle of perfluor；B: after the injection CCR2 targeting pungent bromine alkane nanoparticle of perfluor, it is seen that there are ischemia-reperfusion damages
The myocardium echo of the myocardial segment (antetheca) of wound is remarkably reinforced (shown in white arrow), and remaining heart without ischemia reperfusion injury
Muscle segment section, which has no, to be remarkably reinforced.The CCR2 targeting pungent bromine nanoparticle of perfluor for confirming that the present invention designs has and ischemia-reperfusion
The ability of the highly expressed CCR2 specific binding of myocardial segment, can be realized the targeted ultrasound to Myocardial Ischemia Reperfusion Injury
Radiography.
With19The magnetic resonance targeted developing effect of F-MRI evaluation each group is simultaneously compared: proposed adoption Siemens
Magnetom Trio Tim 3.0T superconducting magnetic resonance instrument is studied rat to obtain image and data.Rat is with 1.5%
Isoflurane anaesthetizes and keeps 37 DEG C during measurement.Use double frequency19F/1H twin coil (under 3.0T field strength,19F is 120.43MHz,1H is 127.49MHz), use TSE sequence conventional sweep cross section T1WI, T2WI sequence (TSE T1WI:TR 600.0ms, TE
9.9ms, the visual field (FOV) 53.4mm × 53.4mm, the number of plies 20, thickness 3mm, excitation number (NEX) 3, matrix 256.0 × 153.6,
Sweep time 2min 54s；TSE T2WI:TR 3700.0ms, TE 108.0ms, FOV 75mm × 75mm, the number of plies 20, thickness
3mm, layer is away from 0.3, NEX 4, matrix 256.0 × 153.6, sweep time 3min 7s) and 3D 19F/1H UTE-SSFP pulse sequence
Column are (while comprising in ultrashort echo time19F/1H RF excitation is acquired with subsequent FID and synchronous double-core acquisition technique, uses
Equilibrium gradient and the radial reading track of Wong type).MR image quantitative analysis carries out mouse chest using Sygno post-processing work station
The measurement of portion's signal strength and ambient noise (repeatedly chooses the average value that area-of-interest (ROI) is measured and obtained SNR, SNR=
SI/SD, SI are the signal strength of tissue, and SD is the standard deviation of ambient noise) and quantitative analysis MR signal strength (SNR) index,
Data are analyzed using MATLAB, and compare the myocardial signal intensity and SNR of each group rat.During the experiment to being targeted in MR body
Photographic parameter carries out reasonably optimizing.
Fig. 6 shows magnetic resonance development (MRI) situation of the pungent bromine alkane nanoparticle of perfluor, wherein A: deionized water；B: deionization
Water+Avidin；C: the pungent bromine alkane nanoparticle of biotinylation perfluor；The pungent bromine alkane nanoparticle+Avidin of D biotinylation perfluor.Illustrate this
There is the pungent bromine nanoparticle contrast agent of the perfluor of invention magnetic resonance to enhance developing function, and it is aobvious that ultrasound-magnetic resonance bimodal may be implemented
Shadow.
Rat is put to death, heart row frozen section is taken out, is received using the confocal microscope observation pungent bromine alkane of each group perfluor
The grain of rice fluorescence display intensity and is compared in ischemic area.
Fig. 7 shows that Ischemia and Reperfusion in vivo in Rats phantom heart frozen section fluorescence microscopy under the microscope (x100), shows antetheca
Visible a large amount of PFOB lipid particles assemble agglomerating (1A) in cardiac muscle, issue bright green fluorescence (1B) and red fluorescence (1C)；
And have no that PFOB lipid particles assemble (2A) in side, rear wall cardiac muscle, also have no sending green and red fluorescence (2B, 2C).Frost
Slice results showed that perfluor of the invention pungent bromine nanoparticle contrast agent only in rat model, there are the hearts of ischemical reperfusion injury
There is aggregation and combines in muscle segment section (antetheca), and does not occur to assemble fixation phenomenon in other myocardial segments (side, rear wall).It is confirmed that
The targets identification function of the pungent bromine nanoparticle contrast agent of perfluor of the invention to myocardial ischemia-reperfusion injury.
Use above specific example is expounded the present invention, is merely used to help understand the present invention, not to
The limitation present invention.The design of those skilled in the art according to the present invention can also be made and several simply push away
It drills, deform or replaces.These are deduced, deformation or alternative are also fallen into scope of the presently claimed invention.
Claims (10)
1. a kind of ultrasound and magnetic resonance bimodal targeted nano granule contrast agent, which is characterized in that the targeted nano granule contrast agent
Including lipidic shell and the pungent bromine alkane core of perfluor being embedded in the lipidic shell, the lipidic shell outer surface is connected with anti-
The monoclonal antibody of destination protein.
2. targeted nano granule contrast agent according to claim 1, which is characterized in that the lipidic shell includes distearyl acid
Phosphatldylcholine, distearyl acid phosphatidyl ethanolamine-polyethylene glycol, distearoylphosphatidylglycerol and cholesterol.
3. targeted nano granule contrast agent according to claim 2, which is characterized in that the distearyl acid phosphatldylcholine,
Distearyl acid phosphatidyl ethanolamine-polyethylene glycol, distearoylphosphatidylglycerol and cholesterol molar ratio is 8.5:(0.5-
1):(0.5-1):(0.5-1)；Preferably, the distearyl acid phosphatldylcholine, distearyl acid phosphatidyl ethanolamine-poly- second two
The molar ratio of alcohol, distearoylphosphatidylglycerol and cholesterol is 8:0.5:0.5:1.
4. targeted nano granule contrast agent according to claim 3, which is characterized in that the anti-destination protein monoclonal antibody
The lipidic shell outer surface is connected to by biotin-avidin-biotin bridging.
5. targeted nano granule contrast agent according to claim 4, which is characterized in that the anti-destination protein monoclonal antibody
To be coupled the anti-destination protein monoclonal antibody of biotinylation for having the first biotin, the distearyl acid phosphatidyl ethanolamine-is poly-
Ethylene glycol is distearyl acid phosphatidyl ethanolamine-polyethylene glycol-biology that wherein polyalkylene glycol moiety coupling has the second biotin
Element, first biotin and second biotin are connected by Avidin, connect the anti-destination protein monoclonal antibody
It is connected to the lipidic shell outer surface.
6. targeted nano granule contrast agent according to claim 5, which is characterized in that the polyethylene glycol is polyethylene glycol
1000, polyethylene glycol 1500, polyethylene glycol 2000, Macrogol 4000 or Macrogol 6000；Preferably, the polyethylene glycol
For polyethylene glycol 2000.
7. targeted nano granule contrast agent according to claim 1 to 6, which is characterized in that the destination protein is
CC class chemokine receptors 2 (CCR2), the anti-destination protein monoclonal antibody are anti-CCR2 monoclonal antibody.
8. a kind of method for preparing ultrasound with magnetic resonance bimodal targeted nano granule contrast agent, which is characterized in that including following step
It is rapid:
(1) it is 8.5:(0.5-1 by molar ratio): (0.5-1): distearyl acid phosphatldylcholine, the distearyl acid phosphide of (0.5-1)
The organic solvent in reaction vessel is added in acyl ethanol amine-polyethylene glycol-biotin, distearoylphosphatidylglycerol and cholesterol
In, dissolution forms clear solution；
(2) it is evaporated in vacuo the clear solution, to form thin film in the reaction vessel；
(3) aqueous buffer is added in the reaction vessel, the film aquation is formed into mother liquor；
(4) the pungent bromine alkane of perfluor is added dropwise to the mother liquor under stiring, obtains mixed liquor；
(6) mixed liquor is added in liposome extruder, the nanopore membrane through the liposome extruder obtains biology
The pungent bromine alkane nanoparticle of elementization perfluor；
(7) excessive Avidin is added in the pungent bromine alkane nanoparticle of Xiang Suoshu biotinylation perfluor, is incubated for 30-60min at 0-4 DEG C,
Centrifugation obtains the pungent bromine alkane nanoparticle-Avidin compound of biotinylation perfluor；
(8) the excessive anti-purpose egg of biotinylation is added in the pungent bromine alkane nanoparticle of Xiang Suoshu biotinylation perfluor-Avidin compound
White monoclonal antibody, is incubated for 30-60min at 0-4 DEG C, and centrifugation obtains targeting the ultrasound and magnetic resonance of the destination protein
Bimodal targeted nano granule contrast agent.
9. preparation method according to claim 8, which is characterized in that the distearyl acid phosphatldylcholine, distearyl acid
Phosphatidyl ethanolamine-polyethylene glycol-biotin, distearoylphosphatidylglycerol and cholesterol molar ratio is 8:0.5:0.5:1,
The organic solvent is chloroform, and the aqueous buffer solution is phosphate buffer, and the polyethylene glycol is cetomacrogol 1000, gathers
Ethylene glycol 1500, polyethylene glycol 2000, Macrogol 4000 or Macrogol 6000, it is preferable that the polyethylene glycol is poly- second
Glycol 2000.
10. preparation method according to claim 8 or claim 9, which is characterized in that the destination protein be CC class chemotactic factor (CF) by
Body 2 (CCR2), the anti-destination protein monoclonal antibody are anti-CCR2 monoclonal antibody.
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